Abstract In this study 41 yeast isolates were isolated from petroleumcontaminated soil samples at petrol stations in the Aseer area, Saudi Arabia. Out of these, nine yeast isolates (KKU-A5, KKU-A6, KKU-A12, KKU-A20, KKU-A23, KKU-A24, KKU-A26, KKU-A29 and KKU-A38) were selected based on their utilization of benzene, toluene and xylene as a sole carbon and energy source. Based on the growth rate, all selected strains showed a high ability for toluene degradation, on the other hand, these isolates had no ability for benzene degradation as well as low ability to degrade xylene except KKU-A29 and KKU-A38 showed no ability to degrade xylene compound. The high growth rates on 15% (v/v) toluene for the nine selected yeasts were 0.7601±0.0042, 0.9398 ±0.0061, 0.7764 ±0.0188, 0.8601 ±0.0049, 0.8128 ±0.0239, 0.7801 ±0.0186, 0.8911 ±0.0340, 0.8541 ±0.0150 and 0.7801 ±0.0186 OD600 respectively after an incubation period of three days. The percentage of toluene removal determined by HPLC analysis for the isolates KKU-A5, KKU-A6, KKUA12, KKU-A20, KKU-23, KKU-A24, KKU-A26, KKU-A29 and KKUA38 was 39.00%, 92.74%, 94.61%, 95.05%, 91.74%, 91.85%, 97.29%, 88.29%, and 85.30% respectively, within the period. The exact identification of the nine selected isolates was detected on the basis of D1/D2 domain of the 26S rRNA gene amplification and sequence determination. Alignment results and the comparison of 26S rRNA gene sequences of the isolates to 26S rRNA gene sequences available in the GenBank database, as well as the phylogenetic analysis, confirmed the accurate position of the isolates as Rhodotorula lactose KKU-A5, Rhodotorula nymphaeae KKU-A6, Rhodotorula graminis KKU-A12, Rhodotorula minuta KKU-A20, Exophiala dermatitidis KKU-A23, Candida davisiana KKU-A24, Rhodotorula slooffiae KKU-A26, Rhodotorula mucilaginosa KKU-A29 and Rhodosporidium diobovatumh KKU-A38. RAPD-PCR fingerprinting was performed to determine the differentiation at the molecular level within the selected seven toluenedegrading red yeast strains (KKU-A5, KKU-A6, KKU-A12, KKU-A20, KKU-A26, KKU-A29 and KKU-A38) and the results indicate that there is no correlation between the RAPD profile and geographic origin sites that these isolates were collected from.